JP5945142B2 - How to evaluate cosmetics for wrinkle improvement - Google Patents

Description

  The present invention relates to a cosmetic evaluation method. Preferably, the present invention relates to a method for evaluating a cosmetic for a wrinkle improving effect.

  Although wrinkles increase as a phenomenon of skin aging with age, there is a great interest in preventing and improving wrinkles, particularly in women, from the viewpoint of beauty and the like. Wrinkles can be broadly divided into fine wrinkles that are generated by the functional deterioration of the epidermis including the stratum corneum and large wrinkles (or facial expression wrinkles) that are generated by the functional deterioration of the dermis deeper than the epidermis. Wrinkles caused by a decrease in the function of the epidermis including the stratum corneum are mainly caused by a decrease in the moisture retention function of the epidermis due to the effects of aging, ultraviolet rays, low temperature, low humidity, and the like. On the other hand, wrinkles caused by functional deterioration of the dermis are caused by collapse of the elastic structural state of the skin composed of collagen and elastin due to the increase and decrease of matrix metalloproteases (MMPs) due to the effects of aging, ultraviolet rays and mechanical stress. (Patent Document 1, Non-Patent Documents 1, 2, and 3). It is known that fine wrinkles generated in the skin are deeply carved to shift to large wrinkles (Non-patent Document 4), and fine wrinkles and large wrinkles are closely related to each other.

  It is a common recognition for many people that fine wrinkles are likely to occur in dry winters. For prevention and improvement of fine wrinkles, moisturizing with cosmetics, especially lotions, milky lotions, creams, cosmetics, etc. It is considered important to do and to humidify with a humidifier. Cosmetics containing a moisturizing agent such as glycerin or hyaluronic acid are particularly used in cosmetics intended for moisturizing.

  Until now, skin barrier function has been improved or improved by using a rough skin model using dry epidermis as a method for screening candidate substances for enhancing stratum corneum transparency (Patent Document 2) and structural changes of stratum corneum intercellular lipids as an index. A method for screening candidate substances (Patent Document 3) has been known. Although these methods screen candidate substances that improve stratum corneum transparency and skin barrier function, there is no description about screening candidate substances that improve wrinkles.

JP 2003-201229 A JP 2010-172200 A JP 2010-175264 A JP 2010-119428 A

Arch Dermatol Res (2002) 294: 405-410 Cell Moto Cytoskeleton 2000 Jan; 45 (1): 1-9 Matrix Biol. 2001 Nov; 20 (7): 397-406 Skin Research and Technology 2011; 17: 135-140

  Development of cosmetics that can repair wrinkles in the stratum corneum is desired, and a method for evaluating cosmetics that can rapidly and on a large scale implement the ability of candidate cosmetics to improve the wrinkles of the stratum corneum is needed. Has been.

  The present inventors have conducted intensive research on the generation mechanism of fine wrinkles generated by the functional deterioration of the epidermis including the stratum corneum, and the fixing fine wrinkles are the functional deterioration of the stratum corneum and the movement of muscles existing under the skin. Grooves of non-fixing horny wrinkles (defined in this specification as "nokojijiwa" in this specification) caused by the passage of a long time without being fully recovered by bathing, moisturizing with cosmetics, etc. (FIG. 1). Specifically, non-fixing koji wrinkles that occur in the stratum corneum are creases that are attached to the hard stratum corneum in the skin, and the stiff stratum corneum becomes mechanical stress and settles into the skin groove. As a result, it was found that epidermis fine wrinkles deeper than the sawed wrinkles were formed.

  Experience has shown that fine wrinkles are caused by a decline in the function of the epidermis including the stratum corneum and are mainly caused by drying in the winter. Although it is known that fine wrinkles can be repaired and improved by sufficiently moisturizing lotions and emulsions for fine wrinkles, the inner epidermis (granular layer, spiny) is once more than the stratum corneum. If the fine lines are fixed to the base layer and the base layer), even if the moisture is maintained, the fine lines disappear temporarily, although the fine lines disappear temporarily. If these fine wrinkles that have settled in the epidermis are left untreated, they may lead to large wrinkles with structural changes such as collagen and elastin in the dermis behind the epidermis. It is a well-known fact that repair is difficult.

  Conventionally, it has been vaguely recognized that fine wrinkles with structural changes of the epidermis are mild that can be repaired by moisturizing and those that are temporary but not disappeared by moisturizing, and in any case to prevent fine wrinkles Moisturizing is considered necessary, and cosmetics capable of repairing fine lines have not been properly evaluated. According to the study by the present inventors, among the wrinkles accompanied by the structural change of the epidermis, the mild wrinkles that can be repaired by moisturizing are wrinkles that have undergone structural change only in the stratum corneum (tempered wrinkles), and even when moisturizing, Wrinkles that have undergone structural changes in the epidermis including the stratum corneum, granule layer, spiny layer, and basal layer in addition to the stratum corneum (“wrinkles” or “fixing fine wrinkles” in this specification). It was found to be defined as

  As for the sawdust defined in this specification, structural change does not occur in the epidermis and dermis other than the stratum corneum. It is in a state with a crease. If such a crease is left untreated, the crease becomes mechanical stress, causing a structural change in the epidermis including the stratum corneum and shifting to fine lines of fixing ability. In addition, it is known that fine wrinkles shift to large wrinkles over a long period of time (Non-patent Document 4). Therefore, in the development of cosmetics, makeup that can repair the folds of the stratum corneum in the remaining wrinkles It has been found that the development of a material is important for the prevention of fixing fine wrinkles, and thus the prevention of large wrinkles. Since such cosmetics can relieve the mechanical stress caused by koji wrinkles, by using such cosmetics for a certain period of time, fine wrinkles that have already settled in the epidermis or large wrinkles that have settled in the dermis. The improvement effect can also be exhibited.

  Furthermore, when the present inventors conducted extensive research on the sawdust, it became surprisingly possible to model the sawdust using the horny layer sheet. In other words, the state where the folds are attached to the stratum corneum is a model of the koji crease, which is the state where the folds are attached to the stratum corneum by the movement of the subcutaneous muscles. It has been found that cosmetics capable of properly repairing wrinkles can repair wrinkles, particularly koji wrinkles, and can contribute to the improvement of fixing wrinkles and wrinkles.

  Therefore, the present inventors have found that the wrinkle improvement degree can be measured by infiltrating the creased horny layer sheet into the candidate cosmetic and drying it, and regarding the wrinkle improving effect of the candidate cosmetic. Invented the evaluation method.

More specifically, the present invention relates to:
(1) A step of folding the stratum corneum sheet;
Soaking in candidate cosmetics;
A step of drying; and a step of measuring the degree of improvement of the wrinkle of the stratum corneum sheet;
Evaluation method about wrinkle improvement effect of candidate cosmetics including
(2) The evaluation method according to item 1, wherein the measurement of the degree of improvement in wrinkles is performed visually.
(3) The evaluation method according to item 1, including a step of photographing the stratum corneum sheet before the dipping step in the candidate cosmetic and after the drying step.
(4) The evaluation method according to item 3, wherein the measurement of the degree of improvement of the wrinkle is performed by image processing of the photographed stratum corneum sheet.
(5) The evaluation method according to any one of items 1 to 4, wherein the wrinkle improving effect is an effect of improving the wrinkle.

  Conventionally, it has been necessary to test animal models and actual human skin for wrinkle evaluation, and there have been problems with individual differences, ethical problems, and the number of tests. Then, since the candidate cosmetics can be evaluated using the stratum corneum sheet, large-scale and homogeneous screening can be performed at the time of product development, and product development can be facilitated.

FIG. 1 is a photograph showing the formation of saw lines on the skin of a human face. FIG. 2 is a diagram showing the wrinkle improvement effect in the stratum corneum sheet wrinkle model. FIG. 3 is a diagram showing the state of wrinkles before and after facial expression movement in vivo and the state of wrinkles after applying model lotion immediately after facial expression movement.

As a first aspect, the present invention provides an evaluation method for the wrinkle improving effect of a candidate cosmetic. More specifically, the evaluation method of the present invention includes the following steps:
The step of folding the stratum corneum sheet;
Soaking in candidate cosmetics;
A step of drying; and a step of measuring the degree of improvement of the wrinkle of the stratum corneum sheet;
One or more steps selected from the group consisting of:

  The candidate cosmetic of the present invention refers to a cosmetic having the possibility of having an effect on wrinkle improvement. More specifically, the candidate cosmetics particularly refer to lotions, emulsions, creams, and beauty essences, but are not intended to improve the skin directly, but include all that are applied to the skin. It is intended to include, for example, sunscreens, insect repellents, hair removal agents, hair restorers, shaving lotions and after shaving lotions. From the viewpoint of the effect of improving wrinkles through infiltration and drying into the stratum corneum sheet, it is considered preferable to use an aqueous cosmetic, for example, a skin lotion or a cosmetic liquid.

  The wrinkle improving effect means that wrinkles formed on the skin disappear. From the viewpoint of the present invention, disappearance or attenuation of koji wrinkles that are creases generated in the stratum corneum is preferable, but not limited thereto, fine wrinkles accompanied by structural changes of the epidermis including the stratum corneum, and structural changes of the dermis This refers to the disappearance or attenuation of large wrinkles.

  In the present invention, the stratum corneum sheet refers to a stratum corneum sheet prepared from living skin (prepared stratum corneum sheet) or a stratum corneum sheet obtained by cell culture (hereinafter referred to as a cultured stratum corneum sheet). From the viewpoint of uniform screening, a culture stratum corneum sheet is preferable.

  The origin of the prepared horny layer sheet is not limited, but those prepared from the horny layer in human skin are particularly preferred. More specifically, it may be acquired by an invasive method such as a surgical means.

  Cultured stratum corneum sheet is a sheet of stratum corneum obtained by in vitro culture, and is a stratum corneum sheet or fibroblasts isolated from a cultured and maintained skin tissue collected from human skin. Or fibroblasts differentiated in vitro from stem cells (eg epithelial stem cells, embryonic stem cells, induced pluripotent stem cells), polymers such as collagen, fibrin, polylactic acid, chitin, chitosan, chondroitin sulfate, hyaluronic acid A horny layer sheet obtained from three-dimensional cultured skin obtained by culturing on a culture support composed of a polysaccharide such as The stratum corneum sheet is isolated from the skin tissue maintained in culture using a technique such as treatment with a proteolytic enzyme in physiological saline with controlled calcium ion and magnesium ion concentrations. As the three-dimensional cultured skin, a human three-dimensional cultured epidermis model product commercially available from LabCyte EPI-MODEL (Japan Tissue Engineering Co., Ltd.) is used. Since the cultured stratum corneum sheet obtained from a commercially available three-dimensional cultured epidermis model product has little difference between lots, it is possible to carry out the evaluation method of the present invention using different lots. In order to prevent the difference between them, one culture horny layer sheet can be cut into several sheet pieces and used in the present invention.

  In the present invention, the stratum corneum sheet can be folded by hand, but in order to make a constant crease, a plate having a hinge portion is used and the plate is folded by folding the plate to a certain angle. Can be attached. Any angle can be selected depending on the depth of the intended crease, for example, selected from 30 °, 45 °, 60 °, 75 °, 90 °, 120 °, 135 °, 150 °, 180 ° The

  In the present invention, the stratum corneum sheet is immersed in the candidate cosmetic in a temperature range in which the cosmetic is normally used. The immersion temperature can be preferably set according to the temperature of the area where the candidate cosmetic is used, and is usually in the range of 10 ° C to 40 ° C, preferably 15 ° C to 30 ° C, preferably 20 ° C to 30 ° C. It is. Any time may be selected as the soaking time, but if the soaking time is shortened, candidate cosmetics with higher wrinkle improving ability can be screened. As immersion time, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours , Or more, such as 4 hours, 8 hours, 12 hours, 24 hours, etc. may be selected. When the candidate cosmetic is scheduled to be applied to the skin, the candidate cosmetic applied to the skin usually evaporates in a relatively short time, so a relatively short immersion time of about 10 seconds to 2 minutes is selected. On the other hand, when the candidate cosmetic is applied using a pack or the like, a longer time such as 10 minutes, 20 minutes, 30 minutes to overnight is selected.

  Ingredients that can be included in the candidate cosmetics include water, ethanol, glycerin, polyethylene glycol and other alcohols as the base, glycine, betaine, pyrrolidone carboxylate and other amino acids, fructose, maltitol, mannitol, Sugars such as trehalose, glyceryl oleate, isostearyl glyceryl as surfactants, plant extracts, jojoba oil, camellia oil, olive oil, squalane, etc. as oil components, hyaluronic acid, collagen, ceramide, ascorbic acid, vitamin E as active ingredients And sodium citrate, citric acid, sodium lactate as an excipient, benzoate, sorbic acid, etc. as preservatives, but is not limited to these, Used It may contain any of the components.

  In the present invention, the stratum corneum sheet infiltrated into the candidate cosmetic by dipping is dried after a predetermined dipping time. The drying step may be performed at any temperature and humidity, but is preferably performed at a temperature and humidity at which the candidate cosmetic is expected to be used. For example, the temperature and humidity vary depending on the region where the candidate cosmetic is sold, but are independently 10 ° C. to 40 ° C., preferably 15 ° C. to 30 ° C., more preferably 20 ° C. to 30 ° C., and humidity 30 to 30 ° C. A range of 90%, preferably 40-80%, more preferably 50-60% is selected. From the viewpoint of shortening the screening time, the drying step may be performed at a temperature higher and / or lower than the temperature and humidity at which the candidate cosmetic is normally expected to be used.

  Measurement of the degree of improvement of the wrinkle of the stratum corneum sheet is performed by visually evaluating the degree of disappearance of the wrinkle. For example, using a standard solution, for example, a 10% glycerin aqueous solution, the same as or more than the standard, based on the degree of disappearance of the crease when the crease addition process, the dipping process, and the drying process are performed under the same conditions. It is possible to screen those having disappeared wrinkles as candidate cosmetics having an excellent wrinkle improving effect. On the contrary, if the wrinkle has not disappeared from the reference, it is determined as a candidate cosmetic that does not have a wrinkle improving effect. Here, any solution can be selected as the reference solution. However, it is also possible to use a commercially available cosmetic or cosmetic liquid as the reference solution.

  The measurement of the degree of improvement of the crease can also evaluate the degree of disappearance of the crease by image processing. In that case, in the measurement process of the improvement degree of a wrinkle, you may further include the process of image | photographing a stratum corneum sheet, and the process of performing a binarization process. By performing the binarization process, it is possible to quantify the degree of disappearance of the wrinkles. From the viewpoint of high-throughput screening of many candidate drugs, evaluation by image processing is preferable.

    Hereinafter, the present invention will be described more specifically with specific examples. In addition, this invention is not limited by this.

Example 1: Formation of Nokojiwiwa In one subject, after washing with soap, acclimation was carried out for 10 to 30 minutes in an artificial climate room (temperature 22-24 ° C, humidity 10-45% RH). I photographed the wrinkles in the corners of the eyes (left in Fig. 1). Subsequently, the eyes were strongly closed and opened 10 times every 5 seconds, which was used as facial expression movement. The wrinkles of the corners of the eyes immediately after the facial expression movement were photographed (Fig. 1 right). As shown by the arrows in FIG. 1, it was shown that a warp wrinkle is formed so as to extend the existing wrinkle immediately after the facial expression movement.

Example 2: Screening of candidate drug for improvement of wrinkle A human epidermis remodeling model was purchased from Japan Tissue Engineering Co., Ltd. This is trypsinized to dry the horny layer sheet at 32 ° C. and 60% humidity to obtain a transparent horny layer sheet. This was bent at an angle of 90 degrees and returned to its original shape to form creases in the stratum corneum sheet. In the crease part, the stratum corneum transparency was lowered, and the damaged part was visualized (left photo in FIG. 2). The folded wrinkled horny layer sheet was used for the evaluation of cosmetic methods or cosmetics as a model of koji wrinkles that occur on the skin surface when the skin expands and contracts due to changes in facial expression. Folded wrinkled horny layer sheet at room temperature (23 ° C) with model lotion (10% glycerin, 3% ethyl alcohol, 0.3% HCO60 (polyethylene glycol (60) hydrogenated hardened castor oil ether (Nikko Chemicals Corporation)) ), 0.09% sodium citrate, 0.01% citric acid) or ethanol for 30 minutes, dried at 32 ° C. and 60% humidity for 18 hours, and photographed (FIG. 2). As a result, it was shown that the model lotion is more effective for the repair of wrinkles.

Example 3: Wrinkle formation by facial expression movement in vivo and improvement of wrinkle by application of model lotion In one subject, after washing with soap, an artificial climate chamber (temperature 22-24 ° C, humidity 10-45%) RH) was acclimated for 10 to 30 minutes, and skin replicas were collected according to the method described immediately after acclimation. Next, replicas were collected after having the panel perform facial expression movements (severe crushing action multiple times). Thereafter, model lotion was applied, and 10 minutes later, replicas were collected by the method performed previously. The collected replica was subjected to three-dimensional image analysis using image analysis software created according to the method described in Patent Document 4, and the wrinkle area ratio was calculated. The results are shown in FIG. This experiment also shows that in-vivo experiments show that the koji wrinkle formed by facial expression movement can be improved by applying model lotion, etc. It has been modeled, and the cosmetics selected by the foldable horny layer sheet of the present invention or the cosmetic screening method can actually improve wrinkles in in vivo experiments. It has been shown.

  Thus, normally, in order to investigate the improvement effect of cosmetics on wrinkles, a monitor test using candidate cosmetics over several weeks for monitors of several to tens of people was necessary. By using the folded wrinkled horny layer sheet as a koji wrinkle model, it is possible to easily and simply perform primary screening of candidate cosmetics having an effect of improving wrinkles.

Claims (5)

The step of folding the cultured horny layer sheet;
Immersing the cultured stratum corneum sheet in a candidate cosmetic;
Drying the cultured stratum corneum sheet ; and
Measuring the degree of improvement in I Flip folding of the culture angle layer sheet;
Evaluation method about wrinkle improvement effect of candidate cosmetics including   The evaluation method according to claim 1, wherein the measurement of the degree of improvement in wrinkles is performed visually.   The evaluation method of Claim 1 including the process which image | photographs a culture sheet before an immersion process and a drying process in candidate cosmetics.   The evaluation method according to claim 3, wherein the measurement of the degree of improvement of wrinkles is performed by image processing of a photographed culture sheet.   The evaluation method according to any one of claims 1 to 4, wherein the wrinkle improvement effect is an improvement effect on the wrinkle.